Vitamin D and its analogs in the treatment of tumors and other hyperproliferative disorders

ABSTRACT

Treatment of hyperporliferative disorders (including tumors and psoriasis) by pulse administration of a drug (such as Vitamin D or an analog) that increases blood or tissue levels of Vitamin D. The drug is administered at a sufficient dose to have an anti-proliferative effect, but the pulsed administration of the drug avoids the development of severe symptomatic or life-threatening hypercalcemia. In particular embodiments, avoidance of hypercalcemia (as measured by serum levels of calcium above normal range) is avoided altogether. In a particular example, the drug is calcitriol administered at an oral dose of about 0.5 mcg/kg once a week.

PRIORITY CLAIM

This is a §371 U.S. national stage of PCT/US99/06442, filed Mar. 25,1999, which was published in English under PCT Article 21(2), and claimsthe benefit of U.S. application Ser. No. 60/079,696, filed Mar. 27,1998.

FIELD OF THE INVENTION

This invention concerns the use of Vitamin D and its analogs in thetreatment of tumors and hyperproliferative disorders.

BACKGROUND OF THE INVENTION

Vitamin D is a generic term for a family of secosteroids that haveaffinity for the Vitamin D receptor, and are involved in the physiologicregulation of calcium and phosphate metabolism. Exposure to the sun anddietary intake are common sources of Vitamin D, but deficiencies of thisvitamin can cause rickets and osteomalacia. Supplementation of dairy andother food products has reduced the incidence of Vitamin D deficiencyconditions in modern society, and medical research concerning thisvitamin has turned to its therapeutic effects in a variety ofpathological conditions.

Vitamin D₃ is synthesized in human skin from 7-dehydrocholesterol andultraviolet light. Vitamin D₃, or its analog Vitamin D₂, can be ingestedfrom the diet, for example in fortified milk products. Vitamin D₂ and D₃undergo hydroxylation first in the liver to 25-hydroxyvitamin D, then inthe kidney to 1α,25-dihydroxycholecalciferol (also known as1,25-dihydroxyvitamin D or calcitriol), which is the principalbiologically active form of Vitamin D. The biological production of thisactive form of the vitamin is tightly physiologically regulated.

Vitamin D exerts its calcium regulating activity through both genomicand nongenomic pathways. Although the nongenomic pathways remain poorlycharacterized, the genomic responses are mediated through binding to thenuclear Vitamin D receptor (VDR). The VDR is a ligand-activatedtranscription factor, which binds the Vitamin D₃ response elementcontained within the promoter/enhancer region of target genes. Vitamin Dmaintains calcium levels in the normal range by stimulating intestinalcalcium absorption. When intestinal absorption is unable to maintaincalcium homeostasis, Vitamin D stimulates monocytic cells to becomemature osteoclasts, which in turn mobilize calcium from bones.

Appreciation for Vitamin D's non calcium-related biological activitiesbegan in 1979, with Stumpf's discovery that radioactive Vitamin Dlocalizes to many tissues not associated with calcium metabolism(Science 206:1188-1190, 1979). In 1981, Abe et al. reported that mousemyeloid leukemia cells possessed VDR, and that their exposure to VitaminD led to terminal differentiation (PNAS USA 78:4990-4994, 1981). Sincethen VDR has been described in carcinomas of the prostate, breast,colon, lung, pancreas, endometrium, bladder, cervix, ovaries, squamouscell carcinoma, renal cell carcinoma, myeloid and lymphocytic leukemia,medullary thyroid carcinoma, melanoma, multiple myeloma, retinoblastoma,and sarcomas of the soft tissues and bone.

In vitro assays using 1,25 dihydroxyvitamin D or its analoguesdemonstrated antiproliferative effects in cell lines derived from manymalignancies including adenocarcinomas of the prostate (Molec. and Cell.Endocrinology 126:83-90, 1997; Proc. Amer. Assoc. Cancer Res. 38:456,1997; J. Ster. Biochem. and Molec. Biol. 58:277-288, 1996; Endocrinology137:1551561, 1996; Endocrinology 136:20-26, 1995; Cancer Research54:805-810, 1994; Endocrinology 132:1952-1960, 1993; and AnticancerResearch 14:1077-1081, 1994), breast (Proc. Amer. Assoc. Cancer Res.38-456, 1997; Biochemical Pharmacology 44:693-702, 1992); colon(Biochemical and Biophysical Research Communications 179:57-62, 1991;Archives of Pharmacology 347:105-110, 1993); pancreas (British Journalof Cancer 73:1341-1346, 1996); and endometrium (Journal of Obstetricsand Gynaecology Research 22:529-539, 1996); lung (Anticancer Research16:2953-2659, 1996); myeloid leukemia (PNAS USA 78:4990-4994, 1981);melanoma (Endocrinology 108:1083-1086, 1981); and sarcomas of the softtissues (Annals of Surgical Oncology 3:144-149, 1996) and bone (Journalof the Japanese Orthopaedic Association 69:181-190, 1995).

Studies in animals have shown antiproliferative activity of Vitamin D orits analogues in prostate cancer (Urology 46:365-369, 1994); breastcancer (J. NCl 89:212-218, 1997; Lancet 1:188-191, 1989); squamous cellcarcinoma (Molecular and Cellular Differentiation 3:31-50, 1995);myeloid leukemia (Blood 74:82-93, 1989 and PNAS USA 80:201-204, 1983)and retinoblastoma (Archives of Opthalmology 106:541-543, 1988; Archivesof Opthalmology 106:536-540, 1988). The mechanism of Vitamin D'santiproliferative effects remains unknown, although it has been proposedthat Vitamin D increases synthesis of TGF-β1 and TGF-β2, decreases theexpression of epidermal growth factor receptors, leads todephosphorylation of the retinoblastoma protein, induces cell cyclearrest in G1, perhaps by induction of the cyclin dependent kinaseinhibitors p21(waf1) and p27(kip1), and induces the production ofinsulin-like growth factor binding protein.

The patent literature is replete with attempts to treat tumors withVitamin D compounds. U.S. Pat. No. 4,391,802 disclosed treatingleukemioid diseases with 1α-hydroxy Vitamin D derivatives. The use of1α-hydroxy derivatives with a 17 side chain greater in length than thecholesterol or ergosterol side chains was disclosed in U.S. Pat. No.4,717,721. Additional Vitamin D analogs are described in U.S. Pat. No.4,851,401 (cyclopentano-Vitamin D analogs), U.S. Pat. No. 4,866,048,U.S. Pat. No. 5,145,846 (Vitamin D₃ analogs with alkynyl, alkenyl, andalkanyl side chains), U.S. Pat. No. 5,120,722 (trihydroxycalciferol),U.S. Pat. No. 5,547,947 (fluoro-cholecalciferol compounds), U.S. Pat.No. 5,446,035 (methyl substituted Vitamin D), U.S. Pat. No. 5,411,949(23-oxa-derivatives), U.S. Pat. No. 5,237,110 (19-Nor-Vitamin Dcompounds), U.S. Pat. No. 4,857,518 (hydroxylated 24-homo-Vitamin Dderivatives). Additional Vitamin D analogs are shown in U.S. Pat. Nos.4,804,502; 5,374,629; 5,403,940; 5,446,034; and 5,447,924.

Few attempts have been made to test Vitamin D's antiproliferativeeffects in humans with cancer. Koeffler et al., Cancer Treatment Reports69:1399-1407, 1985, gave 2 mcg of 1,25-dihydroxyvitamin D daily for 8weeks or longer to 18 patients with myelodysplastic syndrome. Eight of18 patients had minor and transient improvements in the peripheral bloodcounts, but by the end of the 12 week study no patient showedsignificant improvement and 4 patients experienced symptomatichypercalcemia. Bower et al., Lancet 337:701-702, 1991, treated 19patients with locally advanced or cutaneous metastatic breast cancerwith topical calcipotriol, a Vitamin D analogue. Three of the 14patients who completed 6 weeks of treatment showed a 50% reduction inthe bidirectional diameter of the treated lesions and one other patientshowed minimal response, however hypercalcemia was a complication of thetreatment. Palmieri-Sevier et al., Am. J. Medical Sciences 306:309-312,1993, reported a case of long term remission of parathyroid carcinomawhich appeared to be induced and maintained by Vitamin D therapy. Rustinet al., Brit. J. Can. 74:1479-1481, 1996, performed a clinical trialwith a continuous dose of calcitriol in patients with ovarian cancer,and again encountered hypercalcemia.

A phase II trial of oral 1,25-dihydroxyvitamin D (calcitriol) in hormonerefractory prostate cancer was reported by Osborn et al., Urol. Oncol.,1:195-198, 1995. Fourteen patients were given a daily oral dose of0.5-1.5 mcg calcitriol, but no significant response was demonstrated,and clinical deterioration was documented in most of the patients.Thirteen of the patients experienced hypercalcemia, which is the mostcommon side effect of treatment with Vitamin D and its analogs. Concernthat hypercalcemic effects of Vitamin D would preclude the achievementof therapeutic, anti-neoplastic serum levels has inhibited the study ofthe use of this vitamin in humans with cancer. It is an object of thisinvention to provide a method of treatment with Vitamin D drugs (such ascalcitriol) that avoids such hypercalcemia, while permitting the use ofthis class of drugs in the treatment of tumors and otherhyperproliferative diseases.

SUMMARY OF THE INVENTION

Vitamin D and its analogs can be administered in accordance with thepresent invention, for the treatment of neoplastic diseases, such as thetypes of tumors mentioned above, which are responsive to treatment withVitamin D drugs. The method can also be used to treat hyperproliferativeskin diseases, such as psoriasis, disorders of keratinization andkeratosis, or disorders of sebaceous glands, such as acne or sebonheicdermatitis. The method includes administering to the subject atherapeutically effective pulsed dose of the Vitamin D drug in asufficient amount to have a therapeutic effect, without inducinghypercalcemia, particularly symptomatic hypercalcemia, for example grade3 or stage 4 hypercalcemia. This treatment is especially effective toallow the use of highly calcemic drugs (such as drugs having a calcemicindex of 0.5 or more) which are often highly effective in the treatmentof tumors and hyperproliferative diseases, but which have been avoidedin the past because of their calcemic side effects. The dosing regimenof the present invention for the first time allows therapeuticallyeffective antiproliferative (and particularly antineoplastic) amounts ofthese drugs to be given without inducing the dangerous side effect oflife-threatening hypercalcemia, while surprisingly having a prolongedtherapeutic specific anti-tumor or general antiproliferative effect.

In a first disclosed embodiment, the Vitamin D drug is administered to asubject having a neoplasm that expresses a Vitamin D receptor, andresponds to treatment with a Vitamin D drug. Particular types of tumorthat respond to such treatment include adenocarcinomas of the prostate,breast, colon, pancreas and endometrium, as well as small cell andnon-small cell cancer of the lung (including squamous, adenocarcinomaand large cell types), squamous cell carcinoma of the head and neck,transitional cell cancer of the bladder, ovarian and cervical (e.g.squamous cell carcinoma) cancer, renal cell carcinoma, myeloid andlymphocytic leukemia, lymphoma, medullary thyroid carcinoma, melanoma,multiple myeloma, retinoblastoma, and sarcomas of the soft tissues andbone. In particular embodiments, the neoplasm is adenocarcinoma of thebreast or prostate.

In yet other specific embodiments, the Vitamin D drug is one that wouldinduce hypercalcemia (particularly symptomatic or life-threateninghypercalcemia) in a subject to which the drug is given atantiproliferative doses. The method would have particular application todrugs that are as calcemic as calcipotriol (calcemic index of about0.005-0.01), 11α-fluoromethyl-1α,25-(OH)₂-D₃ (having a calcemic index ofabout 0.1), and drugs having a calcemic index greater than 0.5, forexample greater than or equal to 1 (the calcemic index of calcitriol).Drugs with which the method is particularly useful are those drugshaving a half-life no greater than about 1 day, for example no greaterthan about 6 hours, when the dose is given as a therapeuticallyeffective dose. These half-lives are sufficiently short that they allowthe blood level to return to non-calcemic levels for a sufficient periodbetween doses so that full osteoclast activation does not occur. Inparticular embodiments, blood levels of calcium return to normal betweendoses. The Vitamin D drug is administered in an amount that raises aserum level of Vitamin D in the subject with a tumor to asupraphysiologic amount for a sufficient period of time to inducedifferentiation or regression of the tumor without causing symptomatichypercalcemia.

For example, where the Vitamin D analog is calcitriol, it can beadministered in a high pulse dose no more than once every three days,for example once a week. Although calcitriol has been used in the pastto treat cancer, dosages of such regimens have been 0.5-1.5 mcg per dayfor prolonged periods of time, which has caused symptomatichypercalcemia. In accordance with some embodiments of the presentinvention, calcitriol is orally administered in a dose of at least 0.12mcg/kg per day (8.4 mcg in a 70 kg person) no more than once every 5 or6 days, for example once a week. In certain examples, the drug isadministered no more than once every three days, or no more than onceper week. Even higher doses of calcitriol are possible using the pulseddose regimen of the present invention, for example administering thecalcitriol orally in a dose of at least about 0.48 mcg/kg per day, forexample 1 mg/kg per day or higher, e.g. 2-3 mg/kg per day, no more thanonce per week. As the dosage of the calcitriol or other Vitamin D drugincreases, the interval between doses can be increased (for example toas long as 7-10 days) to avoid symptomatic hypercalcemia. It hassurprisingly been observed that pulsed increases in the blood level ofVitamin D are sufficient to have an anti-tumor or antiproliferativeeffect for a prolonged period of time (e.g. 10 days), so that the dosingregimen of the present invention can be followed while encountering alowered risk of hypercalcemia.

The invention also includes a pharmaceutical composition comprising aVitamin D drug in a pharmaceutical dosage form containing at least 5micrograms (mcg) of calcitriol, for example 5-100 mcg. The dosage formmay be an oral, intravenous, intramuscular, topical, subcutaneous,transdermal, sublingual, intranasal, intratumoral or other preparation,but in particular disclosed embodiments the pharmaceutical dosage formis an oral dosage form, such as a tablet or capsule.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription of several preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing peak and trough plasma calcitriol levels insubjects who received the indicated dose of calcitriol over four hours.Peak levels (O) were determined at 6 hours after administration, andtrough levels (O) were determined at 48 hours after administration.

FIG. 2 shows a time course of plasma calcitriol levels in a subject whoreceived a 2.0 μg/kg dose of calcitriol.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Definitions

The following definitions will help with an understanding of the termsused in this specification.

A “Vitamin D drug” is a drug that raises the blood or tissue level ofVitamin D, or has an affinity for the Vitamin D receptor, for examplebinding to that receptor with a Relative Competitive Index (RCI) of 0.05or greater, more particularly 5 or greater, for example 5-250. The RCIis indexed to an RCI of 100 for calcitriol. The term also includes anyof the family of secosteroids with antirhichitic activity, such asVitamin D₂ (ergocalciferol) and Vitamin D₃ (cholecalciferol), theirprecursor molecules such as ergosterol(7-dehydro-22-dehydro-24-methyl-cholesterol) and 7 dehydrocholesterol,25-hydroxyvitainin D₃, the 3-hydroxylated dihydrotachysterol₂, the1α-hydroxylated alfacalcidol (1α-hydroxyvitamin D₃) and calcitriol (1α,25-dihydroxyvitamin D₃), as well as the numerous natural and syntheticVitamin D analogs set forth in the attached Appendix 1 (from Bouillonet. al, Endocrine Reviews 16: 200-257,1995).

Vitamin D drugs also include Vitamin D preparations and analogs that arecurrently in clinical use, such as Rocaltrol® (Roche Laboratories),Calcijex®unjectable calcitriol, investigational drugs from LeoPharmaceutical including EB 1089 (24a,26a,27a-trihomo-22,24-diene-1α,25-(OH)₂-D₃), KH 1060(20-epi-22-oxa-24a,26a,27a-trihomo-1α,25-(OH)₂-D₃), MC 1288 and MC 903(calcipotriol), Roche Pharmaceutical drugs that include1,25-(OH)₂-16-ene-D_(3, 1,25)-(OH)₂-16-ene-23-yne-D₃, and25-(OH)2-16-ene-23-yne-D₃, Chugai Pharmaceuticals 22-oxacalcitriol(22-oxa-1α,25-(OH)2-D₃; 1α-(OH)D₅ from the University of Illinois; anddrugs from the Institute of Medical Chemistry-Schering AG that includeZK 161422 and ZK 157202. Appendix 3 provides additional informationabout chemical structure, route of administration and dosing of some ofthese compounds. Vitamin D analogs also include topical preparations ofVitamin D, such as Calcipotriene (Dovonex) and Tacalcitol, used in thetreatmnent of psoriasis.

A “Vitamin D receptor” (or VDR) is a protein transcription factor, forwhich the gene and its product have already been characterized and foundto contain 427 amino acids, with a molecular weight of about 47,000, orvariants thereof The fall length cDNA of the human VDR is disclosed inBaker et al., PNAS, USA 85:3294-3298, 1988.

“Tumor cells that express (or contain) the Vitamin D receptor” are thosetumors that have been shown to contain the Vitamin D receptor, tumorsthat are subsequently shown to contain the receptor (usinginununohistochemical or other techniques), tumor types (such as breastcancer) that have demonstrated a clinical improvement in response totreatment with calcitriol or its analogs or other Vitamin D drugs, andtumors for which there is epidemiologic data demonstrating anassociation between low Vitamin D levels and higher cancer incidence(such as adenocarcinomas of the prostate, breast and colorectum). Thepresence of Vitamin D receptors can be determined by any means known inthe art, such as any of the techniques disclosed in Pike, Ann. Rev.Nut11:189-216, 1991.

“Elevated (or supraphysiologic) level of Vitamin D” refers to a1,25-dihydroxyvitamin D plasma concentration greater than about 0.15 nM(65 pg/ml), or other Vitamin D concentration greater than normal in thelaboratory where the concentration is measured, for example in humans atotal human plasma concentration greater than about 10 ng/ml of25-hydroxyvitanin D (although this and all other normal values can varydepending on the techniques used to measure the concentration).

“Hypercalcemia” refers to a calcium plasma concentration greater thannormal in the laboratory where the concentration is measured, forexample greater than about 10.5 mg/dL in humans (although this and allother normal values can vary depending on the techniques used to measurethe concentration). Hypercalcemia can be broken into grades 0-4, as setforth in Appendix II.

“Symptomatic hypercalcemia” refers to laboratory demonstratedhypercalcemia associated with one of more of the signs or symptoms ofhypercalcemia. Early manifestations of hypercalcemia include weakness,headache, somnolence, nausea, vomiting, dry mouth, constipation, musclepain, bone pain, or metallic taste. Late manifestations includepolydypsia, polyuria, weight loss, pancreatitis, photophobia, pruritis,renal dysfunction, aminotransferase elevation, hypertension, cardiacarrhythmias, psychosis, stupor, or coma. Ectopic calcification has beenreported when the calcium-phosphate product (multiplying theconcentrations of calcium and phosphate) exceeds 70. “Severe symptomatichypercalcemia” refers to grade 3 or grade 4 hypercalcemia.

A “tumor” is a neoplasm, and includes both solid and non-solid tumors(such as hematologic malignancies). A “hyperproliferative disease” is adisorder characterized by abnormal proliferation of cells, andgenerically includes skin disorders such as psoriasis as well as benignand malignant tumors of all organ systems. “Differentiation” refers tothe process by which cells become more specialized to perform biologicalfunctions, and differentiation is a property that is totally orpartially lost by cells that have undergone malignant transformation.

A “therapeutically effective dose” is a dose which in susceptiblesubjects is sufficient to prevent advancement, or to cause regression ofthe disease, or which is capable of relieving symptoms caused by thedisease, such as fever, pain, decreased appetite or chachexia associatedwith malignancy.

A “pulse” dose of a Vitamin D drug refers to administration of the drugin a sufficient amount to increase the blood or tissue level of theVitamin D drug to a supraphysiologic concentration for a sufficientperiod of time to have a therapeutic benefit, but with a sufficientperiod between doses to avoid hypercalcemia, given the pharmacologicalhalf life of the drug, its rate of elimination from the body, and itscalcemic index.

The “calcemic index” of a drug is a measure of the relative ability of adrug to generate a calcemic response, for example as measured andreported in Bouillon et al., Endocrine Reviews 16:200-257, 1995. Acalcemic index of 1 corresponds to the relative calcemic activity ofcalcitriol. A calcemic index of about 0.01 corresponds to the calcemicactivity of calcipotriol. A calcemic index of 0.5 would correspond to adrug having approximately half the calcemic activity of calcitriol. Thecalcemic index of a drug can vary depending on the assay conducted, e.g.whether measuring stimulation of intestinal calcium absorption (ICA) orbone calcium mobilizing activity (BCM), as reported in Hurwitz et al.,J. Nutr 91:319-323, 1967 and Yamada et al., Molecular, Cellular andClinical Endocrinology (Berlin), pages 767-774, 1988. Hence relativecalcemic activity is best expressed in relation to the calcemic activityof calcitriol, which is one of the best characterized Vitamin D drugs.

Vitamin D Drugs

Normal serum levels of 1,25-dihydroxyvitamin D range between 0.05 and0.16 nM, however therapeutic drug levels necessary for cancer inhibitionhave not been well defined. Skowronski et al. (Endocrinology 136-20-26,1995) demonstrated measurable growth inhibition of LNCaP human prostatecancer cells in vitro at a 1,25-dihydroxyvitamin D concentration of 0.1nM and 50% growth inhibition at a 1.0 nM concentration. Peehl et al.(Cancer Research 54:805-810, 1994) incubated human prostate cancer cellsin primary culture with 1,25-dihydroxyvitamin D concentrations rangingbetween 0.025 and 25 nM and demonstrated half maximal growth inhibitionat levels between 0.25 and 1.0 nM. Previous clinical trials of Vitamin Din the treatment of cancer have proceeded on the assumption that highlevels of the drug were needed for a prolonged period of time to have atherapeutic benefit. The inventors of the present invention, however,have surprisingly shown that intermittent supraphysiologic levels of1,25-dihydroxyvitamin D (for example greater than or equal to 0.25 nM)are sufficient to inhibit cancer growth and other proliferative diseasein mammals. This surprising finding now permits the therapeutic benefitsof Vitamin D therapy to be achieved without substantial risk ofmorbidity from iatrogenic hypercalcemia induced by the therapy.

Calcitriol is a short acting preparation of 1,25-dihydroxyvitamin D,which therefore offers an opportunity for intermittent treatment aimedat achieving high serum 1,25-dihydroxyvitamin D levels for brief periodsof time. This regimen has surprising anti-tumor activity, whileminimizing toxicity, such as hypercalcemia. Calcitriol has primarilybeen studied when chronically administered as replacement therapy, forwhich its usual dose is 0.25-1.0 mcg per day. Peak serum concentrationis reached at 2 hours and serum half life is 3-6 hours. Intestinalabsorption of calcium begins to increase 2 hours after administration.Hypercalcemic effect is maximal at 10 hours and lasts 3-5 days.

In one embodiment of the invention, a sufficient dose of calcitriol isadministered to raise serum 1,25-hydroxyvitamin D levels to atherapeutically effective level for a pulsed dose that has ananti-proliferative effect without causing significant hypercalcemia (forexample symptomatic grade 3 or grade 4 hypercalcemia). With calcitriol,an example of such a dose would produce a serum level of at least about0.5 nM, for example about 0.9 nM or more (e.g. 1-25 nM, for example 5-10nM), for at least 2 hours (e.g. 2-5 hours) and preferably no more than 6hours. In particular embodiments, the pulsed dose of calcitriol does notexceed a dose at which symptomatic hypercalcemia occurs, or morepreferably a pulsed dose at which even laboratory hypercalcemia occurs.

Information about short term effects of higher than replacement doses ofcalcitriol is available for helping predict drug effects. Papapoulus etal., (Clinical Science 62:427429, 1982) gave 2 mcg of calcitriol as asingle oral dose to healthy volunteers and achieved peak1,25-dihydroxyvitamin D sernm concentrations of 0.235 and 0.351 nM.Mason et al. (BMJ 1980:449-450) gave a single oral dose of 4 mcgcalcitriol to healthy volunteers and achieved peak 1,25-dihydroxyvitaminD serum concentrations of 0.42 nM with no elevation in serum calcium.Brickinan et al. (Am. J. Med. 57:28-33, 1974) treated normal volunteerswith calcitriol doses up to 2.7 mcg/day for 7 to 15 days. While calciumabsorption and excretion were increased, no significant elevations inserum calcium were observed. Adams et al. (Kidney Int. 21:90-97, 1982)treated normal volunteers with up to 3 mcg/day of calcitriol for 6-12days and achieved stable 1,25-dihydroxyvitamin D serum levels of0.184-0.235 nM. None of the patients who were on a low calcium dietexperienced elevation in serum calcium. Geusens et al. (Calcifed TissueInt. 49:168-173, 1991) gave 4 mcg of calcitriol per day for 4 days to 27postmenapausal women with osteoporosis or osteoarthritis. Theydemonstrated increased urinary calcium excretion but no increase inurinary hydroxyproline excretion. Four of the 27 patients had a serumcalcium above 10.8 but no clinically significant hypercalcemia wasobserved.

Antiproliferative levels of 1,25-dihydroxyvitamnin D can be achieved forshort periods of time with minimal adverse effects, particularly ifhypercalcemia during short course 1,25-dihydroxyvitamin D therapy isprimarily mediated by increases in intestinal calcium absorption (slowercalcium elevation) rather then osteoclast activation (which can rapidlymobilize calcium from bone). Higher 1,25-dihydroxyvitamin D levels areachievable when the drug is given in conjunction with a reduced calciumdiet to minimize intestinal calcium absorption, and adequate hydrationto maximize calcium excretion. The maximal tolerated dose of calcitriol,when given intermittently has not been defined, but doses as high as0.48 mcg/kg have been tolerated without hypercalcemia.

Higher doses of a Vitamin D drug, sufficient to achieve therapeuticantiproliferative levels, may also be achieved by administering the drugin conjunction with bisphosphonate osteoclast inhibitors, such aspamidronate. Selby et al. (Endocrinology 108:1083-1086, 1981) providedan example of treating hypercalcemia due to Vitamin D with pamidronate.The potential for achieving high serum 1,25-dihydroxyvitamin D levelswhen osteoclasts are inhibited in patients with osteopetrosis ispossible with calcitriol doses as high as 32 mcg/day for 3 months (Keyet al., NEJM 310:409415, 1984) where stable serum levels of1,25-dihydroxyvitamin D peaked at 2.32 nM with no hypercalcemia.

The following Examples illustrate the general method of the presentinvention, as well as specific case histories to illustrate its use.These Examples also provide a general framework for evaluating otherVitamin D drugs, and determining a therapeutically effective dose of aVitamin D drug in a subject with a Vitamin D responsivehyperproliferative disease, without inducing symptomatic iatrogenichypercalcemia.

EXAMPLE 1 General Treatment Plan

A patient with a known Vitamin D receptor positive tumor (such asadenocarcinoma of the prostate, breast, lung, colon or pancreas, ortransitional cell carcinoma of the bladder, or melanoma) may be placedon a prescribed reduced calcium diet prior to treatment, to helpminimize intestinal absorption and allow even higher doses of theVitamin D drug to be used. This reduced calcium diet may be continuedfor the duration of treatment, and for one week after the last dose ofthe Vitamin D drug. The diet ideally restricts daily calcium intake to400-500 mg, by avoiding all dairy products, as well as sardines andother fish canned with their bones, legumes, greens, and any calciumfortified foods or drinks. The subject is then asked to drink 4-6 cupsof fluid more than usual intake starting 12 hours before treatment andcontinuing for days 1, 2, and 3, to assure adequate oral hydration.Magnesium containing antacids, oral calcium supplements, cholestyramine,colestipol, and other bile resin binding agents may also be avoidedduring treatment.

Baseline laboratory tests that may be obtained include serum levels ofcalcium, phosphate, and 1,25-dihydroxyvitamin D. At the initial doselevel, e.g. calcitriol 0.06 mcg/kg po (or another Vitamin D drug forwhich the dose is to be determined) is divided into 4 doses, and one ofthose four doses is taken during each hour for 4 hours until the total0.06 mcg/kg dose is taken. Alternatively, a single higher doseformulation may be ingested. The doses may be rounded up to the nearest0.5 mcg. The subject is monitored daily for symptoms of hypercalcemiafor at least 2-3 days following administration.

The patient may have a variety of laboratory tests performed to monitorthe presence of hypercalcemia, or any physiological consequences ofhypercalcemia. Such tests may include calcium at 0, 24, 48 hours, andbaseline levels of creatinine, total billirubin, ALT, alkalinephosphatase, and a complete blood count. Other possible laboratory testsinclude phosphate, 1,25-dihydroxyvitamin D levels at 0, 6, 24, 48 hours,and 24 hour urine collection for calcium and hydroxyproline on day 2.Subjects are treated with the once a week pulse dose of Vitamin D weeklyuntil disease progression or 4 weeks, whichever comes first, and arefollowed for 2 months from enrollment. If Grade 3 toxicity isencountered, the treatment is stopped.

An initial dose may be chosen from safe doses documented in theliterature, followed by a multistage escalation scheme, such as the onedescribed by Gordon and Willson (Statistic in Medicine 11:2063-2075,1992). Patient accrual occurs in stages I, II, and III. The stagesrequire the accrual of one, three, or six patients respectively beforedose escalation. All patients enrolled at a dose level complete 4 weeksof treatment before the dose level is escalated. In stage I, a singlepatient is entered at each dose level. Accrual continues in stage Iuntil the first Grade 3 toxicity is encountered. When a Grade 3 toxicityis encountered, two more patients are accrued at the same dose level andaccrual will continue in stage II. Doses are reduced one level if oneGrade 4 or 5 toxicity is encountered in stage I.

Accrual continues in stage II if no Grade 3 toxicities are encountered.When one or two Grade 3 or Grade 4 toxicities are encountered, threeadditional patients are accrued at the same dose level and accrualcontinues in stage III. Doses are reduced one level if one Grade 5 orthree Grade 3 or Grade 4 toxicities are encountered in stage II. Instage III six patients are enrolled at each dose level. If only oneGrade 3 toxicity is encountered, the dose will be escalated and theaccrual will revert to stage II. If two or more Grade 3 or greatertoxicities occur, no finther escalation will occur. The MTD (maximumtolerated dose) is defined asthat dose at which ⅓ or fewer of thesubjects experience grade 3 toxicity. For calcitriol, the initial dosewas 0.06 mcg/kg po over 4 hours. At each successive level, the dose isdoubled until the first grade 3 toxicity is encountered. After that,each dose increase is 1.33× of the preceding level according to amodified Fibonacci scheme (Dillman and Koziol, Molecular Biotherapy4:117-121, 1992).

For calcitriol, the pulse dose was given to each subject weekly, and thesubject was monitored for early signs and symptoms of hypercalcemia,such as weakness, headache, somnolence, nausea, vomiting, dry mouth,constipation, muscle pain, bone pain, metallic taste. The patient wasalso monitored for any more serious manifestations, such as polydypsia,polyuria, weight loss, pancreatitis, photophobia, pruritis, renaldysfunction, aminotransferase elevation, hypertension, cardiacarrhythmias, psychosis, stupor, coma, and ectopic calcification.Appropriate treatment is instituted for any patient who demonstrateshypercalcemic toxicity, and the calcitriol is stopped until seruncalcium returns to normal.

The following Table I illustrates a protocol that can be followed witheach drug to determine a tolerated pulse dose. A protocol fordetermining a therapeutic dose will be described in Example 2.

TABLE 1 Example of Protocol for Determining Tolerated Dose WEEKS 5-8WEEKS 1-4 Follow-Up Premature EVALUATION Day Day Day Week Week Until Canl After Ca nl Evaluation & Procedures Screen 1 2 3 5 7 daily every 2Informed Consent x Inclusion / Exclusion Criteria x Physical Exam x x xx Sitting Vital Signs x x x Adverse Effects Recorded x x x x x xCalcitriol administered x Calcium x x x x x x x x Phosphate x x x x x1,25-dihydroxyvitamin D level x xx x x x Creatinine x x x TotalBilirubin x x x ALT x x x Alkaline Phosphatase x x x Albumin x CompleteBlood Count x x β-hCG (select patients) x Urine Collection x x DietQuestionnaire x Tumor measurements (when x x appropriate)

EXAMPLE 2 Determination of Therapeutically Effective Dose

Tumor markers, such as PSA, CA 15-3, and others can be used to assesstumor progression or regression, although the results of such assays cansometimes be difficult to interpret because administration of Vitamin Dhas been shown to increase tumor marker production while inhibitingcancer cell growth. This effect is presumably due to the differentiationinducing properties of Vitamin D.

Alternative means for determining a therapeutic response can also beemployed, for example direct radiographic measurement of tumor lesions.A measurable lesion may be considered one that is bidimensionallymeasurable, with clearly defined margins on physical exams, x-ray, orscan. At least one diameter is preferably greater than 0.5 cm. Bonelesions are not included.

Evaluable disease includes unidimensionally measurable lesions, masseswith margins not clearly defined, palpable nodal disease, lesions withboth diameters less than 0.5 cm, and bone disease. Non-evaluable diseaseincludes disease manifested by pleural effusions, ascites, or diseasedocumented by indirect evidence only (e.g., by lab values which fallinto a category of not being evaluable). The objective status isrecorded at entry into the trial and during week 7 (where week 1 is theweek during which the first dose of the Vitamin D drug is given). If anorgan has too many measurable lesions to measure at each evaluation, aspecific number (such as three lesions) are selected to be followedbefore the patient is entered in the study.

A complete response (CR) is the complete disappearance of all measurableand evaluable disease, with no new lesions. If the subject haseffusions, ascites or disease assessable by surgical restaging (e.g.,testicular and extragonadal gem cell cancer), the disease must becytologically negative. Patients with markers or indirect evidence ofinvolvement must have normalization of abnormal values. All measurable,evaluable and non-evaluable lesions and sites must be assessed. Apartial response (PR) is found in subjects with at least one measurablelesion with 50% decrease of perpendicular diameters of all measurablelesions, with no progression of evaluable disease, and no new lesions.All measurable and evaluable lesions and sites must be assessed. In lungcancer, a greater than 50% decrease in estimated area of evaluable, butnon-measurable, tumor mass, as agreed upon by two independent observers,not to include pleural effusions. Stabilization is a response that doesnot qualify as a complete response, partial response or progression.

EXAMPLE 3 Treatment of Breast Cancer

In this example, a 42 year old woman with breast carcinoma metastatic tonumerous sites in the skeleton received a dose of 11 mcg of calcitriol(Rocaltrol, Roche) administered as 2-2 tablets (0.5 mcg each tablet)divided into four nearly equal doses given in hour one, two, three andfour. The patient received this same therapy on day 1, 8, 15 and 22, andthen was observed on study until day 56, and tolerated the treatmentwell. She had no Grade II or higher toxicities on the NCI toxicitygrading scale (Appendix 2). Subjective beneficial effects observedincluded a reduction in pain and in analgesia required. Objectivebenefits included a progressive decrease in the serum tumor markerCA15-3 from 445 (pre-treatment) to 365 (day 27), 365 (day 48) and 320(day 70). Radiologic evaluation of areas of known bony involvementshowed progressive sclerosis of multiple lesions in the pelvis and righthip, indicating bone healing as a positive response to therapy. No newlesions or pathologic fractures identified were identified by day 64.

EXAMPLE 4 Treatment of Melanoma

In this example a 72 year old man with metastatic malignant melanoma ofthe right jaw received a dose of 37 mcg of calcitriol (Rocaltrol, Roche)administered as 74 tablets (0.5 mcg each tablet) divided into fournearly equal doses given in hour one, two, three and four. The patientreceived this same therapy on day 1, 8, 15 and 22, and observed until atleast day 56. The level of calcitriol in a plasma sample obtained twohours after the last dose of calcitriol (on week one) was determinedusing a commercial assay at Endocrine Sciences, Inc. The level was 1826pg/ml, compared to the range for calcitriol levels in normal controlsbeing 21 to 65 pg/ml. In spite of the markedly elevated levels ofcalcitriol achieved with this weekly schedule, this patient did not haveany subjective or objective toxicity. Levels of serum calcium and otherchemical and hematological parameters in the blood remained normal.

EXAMPLE 5 Summary of Trial Results

Patients

Eligibility criteria included histologically confirmed malignancyrefractory to standard therapy; age ≧18 years; expected survival of >2months; ECOG performance status ≦2; hematocrit ≧30; serum creatinine≦1.2 mg/dL; serum calcium ≦10.5 mg/dL; serum phosphate <4.2 mg/dL; ALT≦60 IUIL; total serum bilirubin <2 mg/dL. Exclusion criteria includedpregnancy, history of hypercalcemia, kidney stones, heart failure orsignificant heart disease including myocardial infarction in the last 3months, known cardiac ejection fraction less than 30%, current digoxintherapy, thiazide diuretic therapy within 7 days, bisphosphonatetreatment within 4 weeks, systemic steroid therapy within 2 weeks, andunwillingness or inability to stop all magnesium containing antacids,bile resin binding drugs, or calcium supplements for the duration of thestudy.

Treatment

Baseline evaluation included a complete history and physical exam,complete blood count, serum creatinine, serum calcium, serum phosphate,total serum bilirubin, ALT, alkaline phosphatase, albumin, serum β-hCGin women of childbearing potential, 24 hour urine collection forcalcium, and tumor measurements.

Patients were asked to maintain a reduced calcium diet for the fourtreatment weeks, with a goal of less than 500 mg per day, as describedin Example 1. Calcitriol (Rocaltrol®, Roche Pharmaceuticals) was givenorally once a week for four weeks. Each weekly dose was given in fourdivided doses given hourly over four hours. The starting dose was 0.06μg/kg.

Monitoring

Complete blood count, serum creatinine, total serum bilirubin, ALT,alkaline phosphatase were monitored weekly. Serum calcium and phosphatewere checked on the treatment day (day 1), and on days 2 and 3.Twenty-four hour urinary calcium excretion was measured on day 2. The1,25-dihydroxyvitaunin D levels were measured by ¹²⁵I radioimmunoassay(Incstar, Stillwater, Minn.) and by a radioreceptor assay using calfthymus 1,25-dihydroxyvitamin D receptor (Endocrine Sciences, CalabasasHills, Calif.). Peak levels were measured two hours after all the pillshad been ingested. Trough levels were measured approximately 48 hourslater.

Compliance with the diet was monitored with a seven day dietary recallquestionnaire directed at calcium rich foods. Daily calcium intacke wasestimated by adding the calcium content of calcium rich foods identifiedby the questionnaire to the estimated calcium content of the basal diet.The calcium content of the basal diet was calculated to be 1 mg ofcalcium/8 Kcal. Caloric intake was estimated with the use of the FoodProcessor 7.0 software (ESHA Research, Salem, Oreg.).

After completing the four week treatment period, patients were monitoredfor four additional weeks. Serum calcium was checked in weeks 5 and 7and tumor measurements were obtained in week 7. All toxicities weregraded according to NCI Common Toxicity Criteria. Response was assessedaccording to WHO guidelines.

Statistical Considerations

The planned dose escalation was governed by the multistage escalationscheme described by Gordon and Willson, 1992. The maximal tolerated dose(MTD) was defined as that dose at which ⅓ or fewer of the patientsexperienced Grade 3 toxicity (64). Patients who had evidence of responseor stable disease, and no Grade 3 or greater toxicity were permitted toreenroll and receive either the same dose or the next higher dose ofcalcitriol. Statistical analysis was performed using StatView 5.0 forWindows software (SAS Institute, Cary, N.C.)

RESULTS

Fifteen different patients were enrolled in 20 cycles of therapy (Table2). Two patients were withdrawn from the study prior to completion ofthe four week regimen because of disease progression. No patientwithdrew because of toxicity of therapy or unacceptability of the diet.Five patients reenrolled for a second cycle of treatment.

TABLE 2 Individual Patients Enrolled on Study Cycle 1 dose Cycle 2 dosePatient Age Gender Malignancy (μg/kg) (μg/kg) 1 79 male Adenocarcinomaof the prostate 0.06 0.12 2 42 female Adenocarcinoma of the breast 0.123 70 male Adenocarcinoma of the lung 0.24 4 72 male Melanoma 0.48 5 53male Squamous Cell of the tonsil 0.48 6 48 female Hepatocellularcarcinoma 0.80 1.60 7 80 male Adenocarcinoma of the prostate 0.96 2.00 853 female Adenocarcinoma of the breast 1.60 9 77 female Adenocarcinomaof the lung 1.92 2.00 10 78 male Adenocarcinoma of the prostate 2.00 1169 male Adenocarcinoma of the prostate 2.00 12 46 female Adenocarcinomaof the breast 2.00 13 47 female Gastrointestinal stromal tumor 2.00 1471 male Adenocarcinoma of the pancreas 2.80 2.80 15 76 maleAdenocarcinoma of the prostate 2.80

No deaths occurred. No patient withdrew from the study due to toxicity,and no Grade 3 or higher toxicity was seen. All observed toxicities arelisted in Table 3.

TABLE 3 Toxicities developed during each treatment course (N = 20)Toxicity Grade 1 Grade 2 Grade 3 Grade 4 Leukopenia  5¹  1² 0 0 Anemia 3 4² 0 0 Thrombocytopenia 2 0 0 0 Hypercalcemia 8 0 0 0 Creatinineelevation 4 0 0 0 Bilirubin elevation 2 0 0 0 ALT elevation 1 0 0 0Alkaline phosphatase elevation 2  1² 0 0 Nausea and vomiting 5 2 0 0Diarrhea 3 1 0 0 Constipation 5 0 0 0 Dyspepsia 4 0 0 0 Headache 5 0 0 0Fever 2 0 0 0 Skin rash 1 0 0 0 Bone or muscle pain 8 0 0 0 ¹All werewithin normal limits of our laboratory (3.4-10.0 k/mm³) but fell intothe Grade 1 toxicity range of 3.0-3.9 k/mm³ ²All had Grade 1abnormalities prior to entry onto study.

The normal range for serum 1,25-dihydroxyvitamin D levels is 0.05-0.16nM(20-65 pg/ml). An approximately linear increase in the peak level wasobserved with increasing dose until the 0.48 μg/kg dose (Table 4, FIG.1). Above this dose, a further elevation of peak levels was not seen.Serum 1,25-dihydroxyvitamin D trough levels returned to normal or nearnormal levels by 48 hours (FIG. 1). A limited study of calcitriolpharmacokinetics showed the expected decay in 1,25-dihydroxyvitamin Dlevels after hour 6 (FIG. 2).

TABLE 4 Mean Peak and 48 hour 1,25-dihydroxyvitamin D levels by doseDose level (μg/kg) Patients Mean peak (nM) Mean 48 hour level 0.06 10.71 0.27 0.12 2 1.10 0.14 0.24 1 2.27 0.21 0.48 2 4.11 0.23 0.80 1 3.530.96 1 3.83 1.60 2 3.65 1.92 1 3.34 2.00 6 4.07 0.26 2.80 2 2.96

Mean serum calcium (normal range 8.5-10.5 mg/dL) increased from 9.55 (SD0.57) mg /dL prior to treatment to 9.76 (SD 0.63) mg/dL 24 hours laterand to 9.88 (SD 0.68) mg/dL at 48 hours (p=0.0002 by a two way repeatedmeasures analysis of variance). All calcium levels above the normalrange returned to normal within 2 days with no intervention. Mean serumphosphate (normal range 2.2-4.2 mg/dL) increased from 3.43 (SD 0.56)mg/dL prior to treatment to 3.98 (SD 0.57) mg/dL 24 hours later anddropped to 3.86 (SD 0.53) mg/dL at 48 hours (p<0.0001 by a two wayrepeated measures analysis of variance). Mean 24 hour urinary calciumexcretion (normal range 100-300 mg) increased from 130 (SD 62) mg with arange of 44-292 mg prior to treatment to 263 (SD 125) mg with a range of59-594 on treatment, measured on day 2 of each treatment week (p<0.0001by a one way repeated measures analysis of variance). There was nostatistically significant increase in urinary calcium excretion duringthe treatment period by the Bonferroni/Dunn test.

Five of eight patients with measurable disease had stable disease. Amongthem, an adenocarcinoma of the lung patient, an adenocarcinoma of thepancreas patient, and a hepatocellular carcinoma patient received twocycles of therapy and remained stable for the entire 16 weeks of theirtime on study. The hepatocellular carcinoma patient had an associated70% decline in her serum AFP level. The remaining three patients withmeasurable disease had evidence of progressive disease.

Four of seven patients without measurable disease had no evidence ofprogression. Among them was the breast cancer patient described inExample 3. A prostate cancer patient received two cycles of therapy, andhad a stable PSA for the entire 16 weeks during which the drug wasadministered, in spite of a rapidly rising PSA prior to enrollment. Theremaining three patients without measurable disease had either tumormarker or clinical evidence of progressive disease.

No patient developed dose-limiting hypercalcemic toxicity fromcalcitriol (<2 mcg/day). Measurements of peak blood calcitriol levels inpatients indicate that blood levels (up to 8.9 nM) are at a level knownto be growth inhibitory for cancer cells in culture. Furthermore, thedrug calcitriol is essentially completely cleared from the blood by day3, and this rapid clearance explains the increased safety profile of theweekly pulse schedule.

An unanticipated result was the finding that escalation of calcitrioldose beyond dose level 0.48 μg/kg did not result in further increases inpeak calcitriol levels. More detailed measurement of calcitriol levelsin one patient (dose level 2.0 μg/kg) indicated that absorption issaturated at high doses rather than delayed, as neither the peak levelsof calcitriol are delayed and the half-life of the drug is not extendedbeyond the usual time observed in lower dose studies. The maximaltolerated dose (MTD) of calcitriol was not determined by the datapresented in this example.

In summary, pulsed weekly administration of calcitriol allowssubstantial escalation of the total weekly dose of calcitriol that canbe administered to patients with advanced malignancies. Peak bloodlevels of calcitriol about 25 fold above the upper limit of normal areachieved with minimal toxicity. These levels are well into the rangewhere antiproliferative effects of calcitriol are observed. Based on theobservation that blood levels of calcitriol do not increase linearlywith increased dose beyond the 0.48 μg/kg level, a dose level of 0.5μg/kg is an example of a dose that is therapeutically effective inpatients whose tumor responds to this therapy, but which does not resultin unacceptable hypercalcemia.

EXAMPLE 6 Preparation of Pharmaceutical Dosage Forms

Preparation of pharmaceutically acceptable compositions of the Vitamin Ddrugs of the present invention can be accomplished using methods wellknown to those with skill in the art. Any of the common carriers such assterile saline solution, plasma, etc., can be utilized with the VitaminD drugs of the invention. Routes of administration include but are notlimited to oral, intracranial ventricular (icv), intrathecal (it),intravenous (iv), parenteral, rectal, topical ophthalmic,subconjunctival, nasal, aural and transdermal. The Vitamin D drugs ofthe invention may be administered intravenously in any conventionalmedium for intravenous injection such as an aqueous saline medium. Suchmedium may also contain conventional pharmaceutical adjunct materialssuch as, for example, pharmaceutically acceptable salts to adjust theosmotic pressure, buffers, preservatives and the like. Among such mediaare polysorbate, normal saline, lactated Ringer's solution, and plasma.Vitamin D is somewhat insoluble, hence solubilizng agents such as sesameoil, or equivalent lipophilic preparations, may be used to administerthe Vitamin D drug.

Embodiments of the invention comprising medicaments, such as tablets orcapsules, can be prepared with conventional pharmaceutically acceptablecarriers, adjuvants and counterions as would be known to those of skillin the art. The medicaments are preferably in the form of a unit dose insolid, semi-solid and liquid dosage forms such as tablets, pills,powders, liquid solutions or suspensions, and injectable and infusiblesolutions, for example a unit dose vial. Effective dosage rangesincluded in the unit dose for calcitriol vary from about 5 mcg to about100 mcg. The unit dosages of the clacitriol are much higher thanpreviously used, because of the unanticipated finding that high pulse,therapeutically effective doses of the drug can be given withoutinducing symptomatic hypercalcemia.

EXAMPLE 7 Determining Binding Affinity

Binding affinity of the Vitamin D drugs for the Vitamin D receptor canbe determined by any acceptable means, such as the VDR binding analysisand Scatchard plots in Peehl et al., Cancer Research 54:805-810, 1994.,which is incorporated by reference.

VDR affinity can be assayed by a competitive receptor assay withradio-labeled calcitriol to determine the Relative Competitive Index(RCI) wherein the RCI for calcitriol is set at 100. The RCI of some ofthe Vitamin D analogs is set forth in Appendix 3.

EXAMPLE 8 Detecting Vitamin D Receptor on Tumor Cells

The presence of the VDR on tumor cells can be detected by the methodsset forth in Peehl et al., which has been incorporated by reference inExample 7. A variety of other assays can be used to detect the VDR,including immunohistochemistry (Kaiser et al., J. Cancer Res. Clin. Onc.122:356-359, 1996); Western blot (Cross et al., Anticancer Research16:2333-2338, 1996); ligand binding assays and DNA probe hybridizationto RNA (Northern blot)(Endocrinology 132:1952-1960); and detection ofRNA by ribonuclease protection assay (Shabahang et al., Annals of Surg.Onc. 3:144-149, 1996).

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

The numerical values in the table for Relative Competitive Index (RCI)for the nuclear VDR and the RCI for the vitamin D binding protein (DBP)tabulate the data for the indicated parameter in relation to the resultfor 1α,25(OH)₂D₃ which is normalized to 100; thus the data valuesrepresent percentages of the 1α,25(OH)₂D₃ value. The key to the dataentries is as follows. The RCI measures the relative ability of ananalog under in vitro conditions to compete with [³H]1α,25(OH)₂D₃ forbinding to the nuclear 1α,25(OH)₂D₃ receptor (VDR) (141) or the plasmavitamin D transport protein (DBP) (142). The cell differentiation dataare related to that for 1α,25(OH)₂D₃ for which the value is set at 1.00.The calcemic index data are set at a value of 1.00 relative to that of1α,25(OH)₂D₃.

SPC indicates the species of origin of the nVDR where c=chick intestine,r=rat intestine, b=bovine thymus, p=pig intestine, m=MCF-7 human breastcancer cells, z=rat osteosarcoma ROS 17/2.8 cells. For the DBP; all dataare obtained from the human protein. The cell differentiation data areobtained in human transformed cell lines where h=HL-60 cells, u=U-937.The calcemic index is a measure of the relative ability of an analog togenerate a “calcemic” response, which is defined differently dependingupon the assay being conducted; c or i=a measure of intestinal Ca²⁺absorption (ICA) or bone Ca²⁺ mobilizing activity (BCM) in the vitaminD-deficient chick (146,553); b=bone resorption; cbp=induction of thevitamin D-induced calcium binding protein, calbindin-D_(28k);sc=elevation senrum Ca²⁺=concentrations; oc=an increase in serumosteocalcin levels; r=an increase in urinary Ca²⁺ excretion. Referencenumbers are indicated in parentheses, and refer to regerence numbers inBoleillon et.al, 1995.

APPENDIX II NCI Common Toxicity Criteria Used to Grade ToxicitiesToxicity Grade 0 1 2 3 4 Blood/Bone Marrow WBC >4.0K 3.0-3.9K 2.0-2.9K1.0-1.9K <1K Platelets WNL 75.0K-WNL 50-74.9K 25.0-49.9K <25K HemoglobinWNL 10.0 g-WNL 8.0-10.0 g 6.5-7.9 g <6.5 g Neutrophils >2.0K 1.5-1.9K1.0-1.4K 0.5-0.9K <0.5K Lymphocytes >2.0K 1.5-1.9K 1.0-1.4K 0.5-0.9K<0.5K Hemorrhage, None Mild, No Gross, 1-2 U Gross, 3-4 U Massive, >4 UClinical Transfusions PRBC PRBC PRBC Infection None Mild Moderate SevereLife-Threatening Gastrointestinal Nausea None Able to Eat IntakeDecreased No Significant Intake Vomiting None 1×/24 hours 2-5×/24 hours6-10×/24 hrs >10×/24 hrs Diarrhea None Increase of 2- Increase of 4-Increase of Increase of 3×/24 hours 6×/24 hours 7-9×/24 hours >10×/24hrs Stomatitis None Painless Ulcers Painful Ulcers, Painful Ulcers,Requires IV Can Eat Cannot Eat Nutrition Hepatic Bilirubin WNL <1.5× WNL1.5-3.0× WNL >3× WNL SGOT/SGPT WNL <2.5× WNL 2.6-5.0× WNL 5.1-20×WNL >20× WNL Alk Phos WNL <2.5× WNL 2.6-5.0× WNL 5.1-20× WNL >20× WNLLiver/Clinical No Precoma Hepatic Coma Change Kidney/Bladder CreatinineWNL <1.5× WNL 1.5-3.0× WNL 3.1-6.0× WNL >6.0× WNL Proteinuria No 1 +2-3 + 4 + Nephrotic Change <0.3 gm % 0.3-1.0 gm % >1.0 gm % SyndromeHematuria Negative Microscopic Gross With Clots Transfusion Alopecia NoLoss Mild Total Cardiovascular Dysrhythmia None Asymptomatic PersistentRequires Therapy Hypotension, V- No Therapy No Therapy tach/V-fibCardiac None Decline of EF by Decline of EF by Mild CHF, Rx RefractoryCHF <20% >20% Responsive Ischemia None Nonspecific ST- AsymptomaticAngina, No Acute MI T Wave changes Ischemic changes InfarctionPericardial None Asymptomatic Pericarditis, rub, Symptomatic TamponadeEffusion EKG changes Effusion Hypertension None Transient, Persistent,Requires Therapy Hypertensive >20 mm Hg >20 mm, No Rx Crisis HypotensionNone Transient, No Fluid Hospitalized Hospitalized Therapy Replacement<48 Hours >48 Hours Pulmonary No Asymptomatic Dyspnea on Dyspnea, noDyspnea at Rest Change Abnormal PFT Exertion exertion Toxicity 0 1 2 3 4Grade Neurologic Neuro-sensory No Mild Paresthesia Moderate Severe Loss,Change Sensory Loss Symptomatic Neuro-motor No Subjective Mild ObjectiveImpairment of Paralysis Change Weakness Weakness Function Cortical NoneMild Moderate Severe, with Coma or Somnolence, Somnolence, Contusion orSeizures Agitation Agitation Hallucination Cerebellar None Slight ChangeSpeech Slur Ataxia Cerebellar Coordination Tremor, Necrosis NystagmusMood No Mild Anxiety or Moderate Severe Suicidal Change DepressionHeadache None Mild Transient, Unrelenting, Moderate-Severe SevereConstipation None Mild Moderate Severe Ileus >96 Hrs Hearing NoAsymptomatic Tinnitus Correctable Loss Deaf, not Change AudiometryCorrectable changes Vision No Symptomatic Blindness Change Subtotal LossSkin No Macular/ Rash with Generalized Exfoliative or Change PapularRash, Pruritus Eruption Ulcerative Rash Asymptomatic Allergy NoneTransient Rash, Urticaria, Mild Serum Sickness, Anaphylaxis Temp <38□ C.Broncho- Bronchospasm spasm, T>38□ C. Fever None 37.1-38□ C. 38.1-40□C. >40□, <24 Hrs >40□, >24 Hrs Local None Pain Inflammation UlcerationPlastic Surgery Phlebitis Rx Weight <5% 5-9.9% 10-19.9% >20% ChangeMetabolic Hyper- <116 116-160 161-250 251-500 >500, GlycemiaKetoacidosis Hypoglycemia >64 55-64 40-54 30-39 <30 Amylase WNL <1.5×WNL 1.5-2.0× WNL 2.1-5.0× WNL >5.1× WNL Hyper- <10.6 10.6-11.5 11.6-12.512.6-13.5 >13.5 Calcemia Hypocalcemia >8.4 8.4-7.8 7.7-7.0 6.9-6.1 <6.0Hypo- >1.4 1.4-1.2 1.1-0.9 0.8-0.6 <0.5 Magnesemia CoagulationFibrinogen WNL .75-1× WNL .5-.74× WNL .25-.49× WNL >24× WNL PT WNL1-1.25× WNL 1.26-1.5× WNL 1.51-2.0× WNL >2.0× WNL PTT WNL 1-1.25× WNL1.2- 1.5× WNL 1.51-2.0× WNL >2.0× WNL

APPENDIX III Vitamin D Analogues Relative Competitive Code index for VDRDosing Analogue Manufacturer Name (Calcitriol = 100) info Comments EB1089 Leo IC 17 (chick) 0.1-0.5 (24a,26a,27a,- Pharmaceutical mcg/kg qdTrihomo-22,24- diene-1□a,25- (OH)₂—D₃) KH 1060 (20- Leo ID 25 (chick) CI= 1.3 Epi-22-oxa- Pharmaceutical 120 (chick) 24a,26a,27a- trihomo-1□,25-(OH)₂—D₃) MC 1288 Leo IE 147 (chick) (1,25(OH₂-20- Pharmaceuticals 120(chick) epi-D₃) MC 903 Leo BT 111 (chick) Rats - 50 Topical form(Calcipotriol) Pharmaceutical 240 (U-937 cells) mcg/kg used in(1□24S-(OH)₂- 76 (chick) qod human breast 22-Ene-26,27- 100 (chick)cancer trial, dehydro-D₃) approved as antipsoriatic CI < 0.011,25-(OH)2-16- Roche HM 165 (chick) Ene-D3 Pharmaceutical 240 (ratintestine) 250 (rat intestine) 1,25-(OH)2-16- Roche V 68 (chick) Mice -0.5 Ene-23-yne-D₃ Pharmaceutical 70 (chick) mcg 3 ×/ 90 (chick) week, 50(rat intestine) 1.6 mcg 80 (chick) qod 25-(OH)2-16- Roche AT 0.4 (chick)Ene-23-yne-D₃ Pharmaceutical <0.4 (chick) 0.07 (rat intestine)22-Oxacalcitriol Chugai EU 15 (chick) CI < 0.001 (22-Oxa-1□,25-Pharmaceutical 12 (chick) (OH)₂-D₃) 100 (rat osteosarcoma) 7 (chick) 100(MCF-7 cells) 1□(OH)D₅ University of — N/A Illinois ZK 161422 (20-Institute of — N/A methyl- Medical (1,25(OH)₂D3 Chemistry - Schering AGZK 157202 (20- Institute of — N/A methyl-23- Medical ene(1,25(OH)₂D3Chemistry - Schering AG 1alpha-(OH)-D3 BP 0.17 (chick)

We claim:
 1. A method for the treatment of a hyperproliferative diseasein a subject, wherein the hyperproliferative disease responds totreatment with a Vitamin D drug, comprising repeatedly administering tothe subject a therapeutically effective pulse dose of the Vitamin Ddrug, no more frequently than once in three days, in a pulse dose ofabout 0.12 mcg/kg per day to about 2.8 mcg/kg per day, each pulsed dosebeing in a sufficient amount to have an antiproliferative effect,without inducing severe symptomatic hypercalcemia in the subject,wherein the Vitamin D drug is 25-hydroxyvit D₃ or1,25-dihydroxycholecalciferol.
 2. The method of claim 1, comprisingadministering the Vitamin D drug to a subject having a neoplasm thatexpresses a Vitamin D receptor, wherein the therapeutically effectivepulse dose is an anti-neoplastic dose.
 3. The method of claim 2, whereinthe neoplasm is selected from the group of cancer of the prostate,breast, colon, lung, head ad neck, pancreas, endometrium, bladder,cervix, ovaries, squamous cell carcinoma, renal cell carcinoma, myeloidand lymphocytic leukemia, lymphoma, medullary thryoid carcinoma,melanoma, multiple myeloma, retinoblastoma, and sarcomas of the softtissues and bone.
 4. The method of claim 3, wherein the neoplasm isbreast cancer or prostate cancer.
 5. The method of claim 1, wherein eachdose of the Vitamin D drug is administered in an amount that raises aserum level of the Vitamnin D drug in the subject with a tumor to asupraphysiologic amount for a sufficient period of time to inducedifferentiation or regression of the tumor without causing symptomatichypercalcemia.
 6. The method of claim 1, wherein the Vitamin D drug iscalcitriol, which is administered in a therapeutically effective pulsedose no more than once in five days.
 7. The method of claim 6, whereinthe calcitriol is administered orally in a dose of at least 0.12 mcg/kgper day no more than once in five days.
 8. The method of claim 6,wherein the calcitriol is administered orally in a dose of at least 0.48mcg/kg or about 1 mcg/kg per day no more than once in a week.
 9. Amethod of treating a tumor in a subject, wherein the tumor expresses aVitamin D receptor and is responsive to treatment with a Vitamin D drug,the method comprising repeatedly administering orally to the subject,once in a three to ten day period, a dose of calcitriol, wherein eachdose of calcitriol is a therapeutically effective dose, of about 0.12mcg/kg to about 2.8 mcg/kg, sufficient to have an antiproliferativeeffect, without inducing severe symptomatic hypercalcemia in thesubject.
 10. The method of claim 9, wherein the calcitriol isadministered to the subject no more than once per week.
 11. The methodof claim 1, wherein the subject eats a reduced calcium diet for asufficient period of time prior to administration of the Vitamin D drugto reduce absorption of dietary calcium.
 12. A method of treating in asubject a tumor that expresses a Vitamin D receptor, the methodcomprising repeatedly administering a dose of a Vitamin D drug to raisea blood level of the Vitamin D drug to a sufficiently supraphysiologiclevel for a sufficient period of time to inhibit growth of the tumor,wherein each dose of the Vitamin D drug is sufficient to inhibit tumorgrowth without inducing hypercalcemia in the subject, wherein theVitamnin D drug is 25-hydroxyvitamin D₃ or 1,25-dihydroxycholecalciferoland the dose is administered no more than once in 3-10 days, and whereinthe Vitamin D drug is admistered in a dose of about 0.12 mcg/kg per dayto about 2.8 mcg/kg per day.
 13. The method of claim 12, wherein theVitamin D drug comprises calcitriol.
 14. The method of claim 13, whereinthe calcitriol is administered in a dose of about 0.50 mcg/kg per dayonce per week.
 15. A method of administering a Vitamin D drug to asubject in need thereof, the method comprising repeatedly administeringtherapeutically effective pulse doses of the Vitamin D drug wherein eachdose is sufficient to achieve intermittent antiproliferative therapeuticlevels of the Vitamin D drug, and wherein the pulse doses areadministered separated by 3-10 days to avoid symptomatic hypercalcemia,and wherein each dose is about 0.12 mcg/kg per day to about 2.8 mcg/kgper day and the Vitamin D drug is 25-hydroxyvitamin D₃ or1,25-dihydroxycholecalciferol.
 16. The method of claim 15, wherein themethod comprises treating a hyperproliferative disorder in the subject.17. The method according to claim 16 wherein the hyperproliferativedisorder is a hyperproliferative skin disease.
 18. The method accordingto claim 17 wherein the hyperproliferative skin disease is selected fromthe group consisting of psoriasis, disorders of keratinization,keratosis, disorders of sebaceous glands, and combinations thereof. 19.The method according to claim 18 wherein the hyperproliferative skindisease comprises disorders of sebaceous glands.
 20. The method of claim19, wherein the hyperproliferative skin disease comprises acne orsebonheic dermatitis.
 21. The method according to claim 1 wherein thehyperproliferative disorder comprises a hyperproliferative skin disease.22. The method according to claim 21 wherein the hyperproliferative skindisease is selected from the group consisting of psoriasis, disorders ofkeratinization, keratosis, disorders of sebaceous glands, andcombinations thereof.
 23. The method according to claim 22 wherein thehyperproliferative skin disease comprises disorders of sebaceous glands.24. The method of claim 19, wherein the hyperproliferative skin diseasecomprises acne or seborrheic dermatitis.
 25. The method of claim 15,wherein each dose of the Vitamin D drug achieves a serum level of about0.5-25 nM of the drug.
 26. The method of claim 25, wherein each dose ofthe Vitamin D drug achieves a serum level of about 1-25 nM of the drug.27. The method of claim 25, wherein each dose of the Vitamin D drugachieves a serum level of about 5-10 nM of the drug.
 28. The method ofclaim 15, wherein the drug is administered in a dose of at least 0.12mcg/kg per day no more than once every 5 or 6 days.
 29. The method ofclaim 28, wherein the drug is administered no more than once a week. 30.The method of claim 29, wherein the drug is administered in a dose of atleast 0.48 mcg/kg per day no more than once per week.
 31. The method ofclaim 30, wherein the drug is administered in a dose of about 2.80mcg/kg per day.
 32. The method of claim 29, wherein the drug isadministered in a dose of about 0.24 to about 2.80 mcg/kg per day. 33.The method of claim 15, wherein the drug is administered orally,intravenously, parenterally, rectally, topically, nasally ortransdermally.
 34. The method of claim 33, wherein the drug is admiteredorally.
 35. The method of claim 15, wherein the drug is administered inconjunction with an other therapeutic agent.
 36. The method of claim 35,wherein the other therapeutic agent is an osteoclast inhibitor.
 37. Themethod of claim 36, wherein the osteoclast inhibitor comprises abiphosphonate osteoclast inhibitor.
 38. The method of claim 15, whereinthe method further comprises administering to the subject an initialdose of the drug, followed by a series of pulsed administrations of anescalated dose, wherein each pulsed administration is in an amountsufficient to have an antiproliferative effect, and wherein eachsubsequent administration follows the preceding administration by aperiod of time sufficient to avoid symptomatic hypercalcemia in thesubject.
 39. The method of claim 15, wherein serum levels of calciumreturn to normal levels of calcemia between doses of the drug.
 40. Themethod of claim 15, wherein the drug would induce hypercalcemia ifadministered daily at the dose at which it is administered in pulsedoses separated by 3-10 days.
 41. A method of administering a Vitamin Ddrug to a subject at an anti-hyperproliferative dose substantiallywithout symptomatic significant hypercalcemia, wherein the methodcomprises repeatedly administering therapeutically effective pulseddoses of the Vitamin D drug, wherein each dose of the Vitamin D drug isa dose that is sufficient to have an antiproliferative effect, the doseis repetitively administered separated by 3-10 days, and the Vitamin Ddrug is calcitriol, and wherein the dose is about 0.12 mcg/kg per day toabout 2.8 mcg/kg per day.
 42. The method of claim 41, wherein each doseof the drug is sufficient to have an anti-tumor antiproliferativeeffect.
 43. The method of claim 42, wherein the dose of the drug issufficient to raise a serum level of the drug to 0.5-25 nM.
 44. Themethod of claim 42, wherein the dose of the drug is about 0.48-2.80mcg/kg.
 45. The method of claim 42, comprising administering the VitaminD drug to a subject having a neoplasm that expresses a Vitamin Dreceptor.
 46. The method of claim 42, wherein the neoplasm is selectedfrom the group of cancer of the prostate, breast, colon, lung, head andneck, pancreas, endometrium, bladder, cervix, ovaries, squamous cellcarcinoma, renal cell carcinoma, myeloid and lymphocytic leukemia,lymphoma, medullary thryoid carcinoma, melanoma, multiple myeloma,retinoblastoma, and sarcomas of the soft tissues and bone.
 47. Themethod of claim 44, wherein the dose is administered orally.
 48. Themethod of claim 41, wherein the doses are separated by 7-10 days. 49.The method of claim 43, wherein the dose of the drug is sufficient toraise the serum level of the drug to 0.5-25 nM for no more than about 6hours.
 50. The method of claim 15, wherein the pulse dose isadministered in divided doses over a one day period.
 51. The method ofclaim 41, wherein each pulse dose is administered in divided doses overa one day period.
 52. The method of claim 15, wherein each pulse dose isadministered as a single dose.
 53. The method of claim 41, wherein eachpulse dose is administered as a single dose.
 54. The method of claim 1,wherein the Vitamin D drug is calcitriol, which is administered in adosage form that contains 5-100 mcg calcitriol.